Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface is dependent on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a serves as an essential component of the melanosome receptor for myosin Va. We have now shown that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null(dilute) phenotype in wild type melanocyes is absoltutely dependent on the presence of Exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue melanosome distribution in dilute melanocytes is absolutely dependent on the presence of Exon F, since melanocyte myosin Va, and melanocyte myosin Va without Exon D colocalize and rescue, while melanocyte myosin Va without Exon F and brain myosin Va do not colocalize or rescue. These results imply that if myosin Va and Rab27a are a motor: receptor pair, their physical association should be Exon F-dependent. Consistent with this, Rab27a present in melanocyte detergent lysates binds to beads coated with purified, full-length, FLAG-tagged melanocyte myosin Va and melanocyte myosin Va lacking Exon D, but not to melanocyte myosin Va lacking Exon F, or to brain myosin Va. Furthermore, the preparation of lysates in the presence of GDP rather than GTP reduces the amount of Rab27a bound to melanocyte myosin Va by ~4 fold. Finally, the stable interaction of myosin Va and Rab27a appears to require at least one additional lysate-derived factor, since purified, GTP-loaded Rab27a does not bind appreciably to myosin Va-coated beads. Together, these results firmly establish that myosin Va and Rab27a function in the context of the melanosome as a motor: receptor pair, show that their interaction requires Exon F and at least one additional protein, and suggest that the recruitment of myosin Va on to the melanosome surface should be regulated by factors controlling the nucleotide state of Rab27a. The Dictyostelium CARMIL protein acts as a scaffold to link capping protein (CP) and the Arp2/3 complex to type I myosins through their SH3 domains. To further characterize this complex, we have purified Acanthamoeba CARMIL to homogeneity. Analytical ultracentrifugation, electron microscopy, and chemical crosslinking studies show that CARMIL is in a monomer: dimer equilibrium with an association constant of ~ 1uM. The most striking observation regarding the purification of CARMIL is that it copurifies extensively with CP. Complete dissociation of the two by gel filtration requires a mild chaotropic agent or low pH (5.4). Purified CP rebinds to CARMIL with a maximum stoichiometry of two CP heterodimers per CARMIL momomer, and with an affinity of ~ 100 nM. Given this affinity, and the cellular concentrations of CARMIL (~2 uM) and CP (~1 uM), CARMIL would be predicted to significantly influence barbed end capping in vivo. Recent in vitro experiments show that the CARMIL: CP complex can cap actin filaments, indicating that CARMIL does not function to simply sequester CP in an inactive pool. Rather, CARMIL and CP form a novel barbed end cap that (i)may be translocated to barbed ends by myosin I, and (ii) may, through its ability to also recruit and activate Arp2/3, allow the assembly of a new actin filament off of a capped end.